Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions

Abstract

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI− mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI− mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.